U.S. patent number 6,251,858 [Application Number 08/591,481] was granted by the patent office on 2001-06-26 for derivatives of oligosides, their process of preparation and their applications.
This patent grant is currently assigned to I.D.M. Immuno-Designed Molecules. Invention is credited to Roger Mayer, Michel Monsigny, Annie-Claude Roche, Nadia Sdiqui.
United States Patent |
6,251,858 |
Monsigny , et al. |
June 26, 2001 |
Derivatives of oligosides, their process of preparation and their
applications
Abstract
The invention relates to compounds comprising one or several
oligosides, each of said oligosides being fixed in a covalent
manner on one or many molecules, matrixs or particles, specifically
one, two or three, via an intermediary molecule possessing a
nitrogen atom carried by a carbon in .alpha. of a C.dbd.O group and
one or many functional groups, specifically one, two or three, the
covalent bond between said intermediary molecule and the oligoside
being done by the intermediary of said nitrogen atom, and the
covalent bond between said intermediary molecule and said molecule,
said matrix, said particle, or said molecules, said matrixs, said
particles being done by the intermediary of the said functional
groups of said intermediary molecule and appropriate functional
groups to the molecule(s), the matrix(ces) or the particle(s).
Inventors: |
Monsigny; Michel
(Saint-Cyr-en-Val, FR), Roche; Annie-Claude
(Sandillon, FR), Sdiqui; Nadia (Orleans,
FR), Mayer; Roger (Orleans, FR) |
Assignee: |
I.D.M. Immuno-Designed
Molecules (Paris, FR)
|
Family
ID: |
9464582 |
Appl.
No.: |
08/591,481 |
Filed: |
February 21, 1996 |
PCT
Filed: |
June 15, 1995 |
PCT No.: |
PCT/FR95/00790 |
371
Date: |
February 21, 1996 |
102(e)
Date: |
February 21, 1996 |
PCT
Pub. No.: |
WO96/00229 |
PCT
Pub. Date: |
January 04, 1996 |
Foreign Application Priority Data
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|
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Jun 23, 1994 [FR] |
|
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94 07738 |
|
Current U.S.
Class: |
530/300; 514/414;
530/322; 530/330; 536/23.1; 536/123 |
Current CPC
Class: |
C07H
19/048 (20130101); C07H 19/044 (20130101); C07H
3/06 (20130101); C07H 21/00 (20130101); A61K
47/61 (20170801); A61P 31/12 (20180101); A61P
35/02 (20180101); C07H 15/12 (20130101) |
Current International
Class: |
A61K
47/48 (20060101); C07H 21/00 (20060101); C07H
15/12 (20060101); C07H 19/00 (20060101); C07H
15/00 (20060101); C07H 19/044 (20060101); C07H
19/048 (20060101); C07H 3/06 (20060101); C07H
3/00 (20060101); A61K 038/16 () |
Field of
Search: |
;530/330,322 ;514/8,414
;536/23.1,123 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2277823 |
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Feb 1976 |
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FR |
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WO 88/00592 |
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Jan 1988 |
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WO |
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WO 93/04701 |
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Mar 1993 |
|
WO |
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WO 94/03184 |
|
Feb 1994 |
|
WO |
|
Other References
Reutter et al., Z. Lebensm.--Unters. Forsch, 188(1), 28-35.
(abstract), 1989.* .
E. Bonfils et al., "Drug targeting: synthesis and endocytosis of
oligonucleotide-neoglycoprotein conjugates", Nucleic Acids
Research, vol. 20, No. 17, Sep. 11, 1992, pp. 4621-4629..
|
Primary Examiner: Jones; Dwayne C.
Assistant Examiner: Delacroix-Muirheid; C.
Attorney, Agent or Firm: Young & Thompson
Parent Case Text
This application is a 371 of PCT/FR95/00790 filed Jun. 15, 1995.
Claims
What is claimed is:
1. Compounds of the general formula (I) ##STR71##
in which:
a=0 or 1,
j=0 or 1,
b=0 or 1,
p=2 to 4,
provided that
a=b=0, when j=1, which leads to the presence of a cyclic
molecule,
or a=b=1, when j=0 which implies the absence of the
(CH.sub.2).sub.p group,
D represents a residue of an organic acid of the formula ECO.sub.2
H, wherein E is H or an alkyl chain of 1 to 10 carbon atoms,
Z represents
B, B being H, an alkyl of 1 to 10 carbon atoms or a side chain of
an .alpha. amino acid, or
B'--P', B' being an alkylene chain of 1 to 10 carbon atoms or a
side chain of an .alpha. amino acid, the said chains containing a
group derived from a functional group able to be activated, P'
having the significations indicated hereafter,
X represents: ##STR72##
m being an integer from 0 to 10, k=0 or 1
Q representing OH, OCH.sub.3, OCH.sub.2 --C.sub.6 H.sub.5,
O--C.sub.6 H.sub.5, O--C.sub.6 F.sub.5, O-pC.sub.6 H.sub.4
--NO.sub.2, or ##STR73##
R representing a group possessing an alcohol, phenol, thiol, or
amine function,
A.sub.i representing an organic radical,
P, P', and P" are identical or different and represent:
a matrix support for affinity chromatography;
a bead of gold, or latex;
a protein;
a lipid;
oligonucleotides; or
a polymer;
P, P' and P" possessing at least one chemical function allowing a
condensation reaction by reaction with an oligopeptide,
wherein the oligosidyl moiety of formula (I) comprises a terminal
cyclic hemiacetal residue in which the hemiacetalic hydroxy is
replaced by the N of formula (I), thereby to render said hemiacetal
non-reducing.
2. Compound according to claim 1, characterized in that P' or P"
represents an oligonucleotide, or R represents fluoresceine or a
derivative thereof or another fluorescent derivative, and P' or P"
represents an oligonucleotide.
3. Compounds according to claim 1, of the general formula (II)
##STR74##
in which:
X represents:
either the group [NH--(A.sub.i)--CO].sub.m --P,
or the group [NH--(A.sub.i)--CO].sub.m --R--P; and
P represents:
a matrix support for affinity chromatography;
a bead of gold or latex;
a protein,
an oside receptor,
a lipid; or
oligonucleotides,
wherein a, j, b, p, D, B, m, A.sub.i, and R are as defined in claim
2.
4. Compounds according to claim 1, of the general formula (III)
##STR75##
in which X represents [NH--(A.sub.i)--CO].sub.m --P or
[NH--(A.sub.i)--CO].sub.m --R--P, and
wherein R, A.sub.i, m, and P are as defined in claim 1.
5. Compounds according to claim 1, of the general formula (IV)
##STR76##
in which X represents [NH--(A.sub.i)--CO].sub.m --P or
[NH--(A.sub.i)--CO].sub.m --R--P, and
wherein R, A.sub.i, D, m, and P are as defined in claim 1.
6. Compounds according to claim 1, of the general formula (V)
##STR77##
in which X represents [NH--(A.sub.i)--CO].sub.m --P or
[NH--(A.sub.i)--CO].sub.m --R--P, and
wherein R, A.sub.i, m, and P are as defined in claim 1.
7. Products of a general formula (Ia) ##STR78##
in which:
a=0 or 1,
j=0 or 1,
b=0 or 1,
p=2 to 4,
provided that
a=b=0, when j=1, which leads to the presence of a cyclic
molecule,
or a=b=1, when j=0 which implies the absence of the
(CH.sub.2).sub.p group,
D represents a residue of an organic acid of the formula ECO.sub.2
H, wherein E is H or an alkyl chain of 1 to 10 carbon atoms,
Z.sub.1 represents
B, B being chosen from among: H, an alkyl chain of 1 to 10 carbon
atoms, and a side chain of an .alpha. amino acid, or
B', B' being chosen from among: an alkylene chain of 1 to 10 carbon
atoms, or a side chain on an .alpha. amino acid, said chains
containing a functional group,
X.sub.1 represents
the group [NH--(A.sub.1)--CO].sub.m --R,
or [NH--(A.sub.1)--CO].sub.m --Q
R representing a group possessing an alcohol, phenol, thiol, or
amine function,
Q representing OH, OCH.sub.3, OCH.sub.2 --C.sub.6 H.sub.5,
O--C.sub.6 H.sub.5, O--C.sub.6 F.sub.5, O-pC.sub.6 H.sub.4
--NO.sub.2, or ##STR79##
m being an integer from 1 to 10,
A.sub.i represents an organic radical,
wherein the oligosidyl moiety of formula (Ia) comprises a terminal
cyclic hemiacetal residue in which the hemiacetalic hydroxy is
replaced by the N of formula (Ia), thereby to render said
hemiacetal non-reducing.
8. Compounds or products according to claim 7, characterized in
that R represents the following radicals: ##STR80##
--NH--pC.sub.6 H.sub.4 --N.dbd.C.dbd.S and its precursors:
--NH--pC.sub.6 H.sub.4 --NO.sub.2 --NH--pC.sub.6 H.sub.4 NH.sub.2
--NH--CH.sub.2 --(CH.sub.2).sub.m --C(.dbd.
NH.sub.2.sup.+)OCH.sub.3 --NH--CH.sub.2 --(CH.sub.2).sub.m --CN
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--CH.sub.2
--(CH.sub.2).sub.m --C(.dbd. NH.sub.2.sup.+)OCH.sub.3
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--CH.sub.2
--(CH.sub.2).sub.m --CN ##STR81##
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--CH.sub.2 --T
T=Br, I, Cl --NH--CH.sub.2 --CH.sub.2 --NH--CO--CH.sub.2
(CH.sub.2).sub.m --S--S--Pyr
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --S--S--Pyr
##STR82##
--NH--(CH.sub.2).sub.m --pC.sub.6 H.sub.4 OH --NH--CH.sub.2
--(CH.sub.2).sub.m --CH.sub.2 --NH--CO--(CH.sub.2).sub.m --pC.sub.6
H.sub.4 OH ##STR83##
9. Compounds according to claim 7, characterized in that the
oligosidyl residue is an oligosaccharide comprised of from 2 to 50
oses and is chosen from:
Three antenna complex of the formula:
simple or complex osides recognized by the lectin membranes, and
chosen from:
a. Asialo-oligoside of lactosamine triantenna: receptor of
asialoglycoprotein ##STR85##
b. Asialo-oligoside of lactosamine tetraantenna: receptor of
asialoglycoprotein ##STR86##
c. Lewis.sup.X : LECAM 2/3 ##STR87##
d. Sialyl Lewis.sup.X : LECAM 3/2 ##STR88##
e. Derivative of Lewis.sup.X sulfate (HNK1): LECAM 1 ##STR89##
f. Oligomannoside: receptor of mannose ##STR90##
g. Phosphorylated oligomannoside: receptor of mannose 6 phosphate
##STR91##
h. Oligosaccharide of sulfated lactosamine receptor of sulfated
GalNAc 4 ##STR92##
10. Process for preparing compounds according to claim 7,
comprising:
(a) condensing an oligoside having a free reducing sugar, on the
nitrogen atom of an intermediary molecule, said nitrogen atom
belonging to an amine group, linked to a carbon atom placed in
.alpha. of a C.dbd.O group, in presence of a catalyst and in a
solvent appropriate for obtaining a derivative of glycosylamine in
which the terminal ose of the oligoside conserves its cyclic
structure, and in which the semiacetalic hydroxyl is replaced by
the .alpha. amine of one of said intermediary molecules,
wherein if the intermediary molecule does not possess a side chain
containing a functional group, acylating the derivatives of
glycosylamine obtained at the end of step (a) by the addition of an
organic acid activated by an activator to obtain a derivative of
N-acylated glycosylamine, or
wherein if the intermediary molecule possesses a side chain
containing a carboxylic group, activating the carboxylic group to
intramolecularly react with said .alpha. amine, leading to a
cyclization inside the intermediary molecule, to obtain a
derivative of N-acylated glycosylamine.
11. The process of claim 10, where in step (a) the intermediary
molecule is selected from the group consisting of: an .alpha. amino
acid, a natural or synthetic, derivative of said .alpha. amino
acid, an amino acid in N-terminal position of a peptide, and a
peptidic derivative.
12. Products according to claim 7, of the general formula (IIa)
##STR93##
wherein a, j, b, p, D, B, m, A.sub.i, and R are as defined in claim
7.
13. Products according to claim 7, of the general formula (IIIa)
##STR94##
wherein A.sub.i, m, and R are as defined in claim 7.
14. Products according to claim 7, of the general formula (IVa)
##STR95##
wherein D, B, m, A.sub.i and R are as defined in claim 7.
15. Products according to claim 7, of the general formula (Va)
##STR96##
wherein A.sub.i, m, and R are as defined in claim 7.
16. Products of a general formula ##STR97##
in which:
B is chosen from among: H, an alkyl chain of 1 to 10 carbon atoms,
and a side chain of an .alpha. amino acid,
R represents a group possessing an alcohol, phenol, thiol, or amine
function,
m is an integer from 1 to 10,
A.sub.i represents an organic radical,
wherein the oligosidyl moiety of formula (VI) comprises a terminal
cyclic hemiacetal residue in which the hemiacetalic hydroxy is
replaced by the N of formula (VI), thereby to render said
hemiacetal non-reducing.
17. Products of a general formula ##STR98##
in which:
P=2 to 4,
R represents a group possessing an alcohol, phenol, thiol, or amine
function,
m is an integer from 1 to 10,
A.sub.i represents an organic radical,
wherein the oligosidyl moiety of formula (VII) comprises a terminal
cyclic hemiacetal residue in which the hemiacetalic hydroxy is
replaced by the N of formula (VII) thereby to render said
hemiacetal non-reducing.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to oligoside derivatives, their process of
preparation, and their applications.
2. Description of the Related Art
Natural oligosides are able to be prepared in free form from
various physiologic liquids such as milk, or extracts from natural
or transformed products (honey, beer, etc.). Natural oligosides are
also able to be obtained by cutting a glycoside bond from one of
the sugar moieties of glycoconjugates (glycolipids, glycoproteins,
polyosides, proteoglycans, etc.), by hydrolysis with the aid of
enzymes or by chemical catalysis from said glycoconjugates.
The natural oligosides are able to be used as substrates, as
inhibitors, as recognition signals, etc. In the majority of cases,
it is advisable to fix the oligoside on a molecule, matrix or
particle, which can be chosen from:
a matrix as a support for affinity chromatography;
a bead of gold or latex, for histology and cytology;
a protein for visualilzation, purification, etc., in particular 1)
specific receptors of osides, receptors which are called lectins,
adhesins, agglutinins, etc., or 2) proteins with or without
enzymatic activity, which have an affinity for the osides, in
particular the glycosyltransferases, exoglycosidases or
endoglycosidases
a lipid for the characterization of the preceding receptors;
oligonucleotides for selectively increasing their capture by
targeted cells;
a protein or polymers for the targeting of drugs, oligonucleotides
or genes, or for obtaining intramolecular inhibitors.
The synthesis of derivatives of oligosides able to be linked by
covalent means to a protein, a matrix, an oligonucleotide, or by
general means to an organic molecule or a particle, in all cases
preserving the integrity of the structure and functions of each of
its sugar components--which is necessary for preserving the
functional capacity of the oligoside can be obtained essentially in
two ways: the total synthesis de novo and the intermediary
transformation into glycosylamine. A third way which leads to an
equally useful derivative but which destroys the terminal
reductorose is amination in a reducing medium.
Concerning total synthesis, this requires a selective protection of
the hydroxyls non-engaged in a glycoside bond, steps of
condensation, steps of selective deprotection, and a step of final
deprotection. Even if over the last decades the yields of some of
these steps were able to be improved, this is a long process and
the overall yield remains modest, and this all the more as the
oligoside to be synthesized is more complex.
The yields for each step are between 20 and 95% according to the
steps considered.
For example, the synthesis of a para-nitrophenyl derivative of a
pentasaccharide such as the determinant of Lewis x:
requires in total several tens of steps. Each step has a yield of
between 50 and 95%, more generally 80%. In total the yield of the
product sought is of some %.
The elevated number of steps arises from the fact that the alcohol
functions of each ose must be protected in a different manner
depending on whether the hydroxyl under consideration will or will
not be implicated in a condensation reaction.
A sugar such as GlcNAc which will be substituted 2 times will
receive momentarily 3 different substitutes in order to permit a
selective substitution on the hydroxyl 3 by galactose, on the
hydroxyl 4 by fucose, the hydroxyl 6 remaining protected until the
final deprotection.
For each step of condensation, the yield is affected by the fact
that the product formed is in general a mixture of the two (.alpha.
and .beta.) anomeric forms.
Moreover, it is necessary to point out that the intermediary
products have to be purified, either by crystallization, or by
chromatography, which contributes significantly to the total
duration of the synthesis. Finally, it should be noted that the
yields may be very weak for certain steps because of steric
hindrance, which is specifically the case at the level of the
branches: 2 sugars substituting a single monosaccharide.
All in all, this global synthesis is very costly and takes a long
time.
In the case of the formation of a glycosylamine followed by
acylation, this way depends on a reaction described at the end of
the last century: an oside possessing a reducing sugar, incubated
in the presence of an elevated concentration of ammoniac, of an
ammonium salt or an aromatic amine, is transformed in a reversible
manner into glycosylamine.
For example, the lactose gives a lactosylamine, with ammoniac:
or with the aniline:
This reaction is however reversible, which is to say the isolated
product, dissolved in a buffer, becomes hydrolyzed leading to give
back the original products.
The glycosylamine may however be stabilized by acylation, for
example by selective N-acetylation:
On these bases, it has recently been proposed to substitute the
amine of oligosylamines by an organic compound possessing a
finctional reactive group. Manger et al., 1992, Biochemistry 31,
10724 and 31, 10733.
This route comprises the following steps:
1) incubation of the oside possessing a terminal reducing sugar in
the presence of a highly concentrated ammonium salt.
For example,
2) Purification of the glycosylamine by chromatography on a column
to eliminate the ammonium salt excess.
3) Substitution of the amine of the glycosylamine by reaction with
an activated derivative of monochloroacetic acid, in alkaline
medium.
For example,
4) Transformation of the chloroacetyl residues into glycyl
residue.
The chloroacetylglycosylamide is incubated in the presence of a
highly concentrated ammonium salt, which allows the introduction of
an amine function. For example:
5) Purification of glycyl-glycosylamide by chromatography on column
to eliminate the ammonium salts.
6) Condensation of the glycyl-glycosylamide and of a compound able
to selectively react with an amine group of the glycyl residue.
For example:
in which G is an activator of the carboxylic function.
This route in 6 steps implies two steps of intermediary
purification and the use of a toxic product: an activated
derivative of chloroacetic acid.
The yield of each step is variable and ranges between 50 and 95%;
the global yield is less than approximately 60%.
Another pathway has also been suggested, but it requires the
transformation of the reducing sugar into polyol; this route was
proposed in 1974 by Gray (Arch. Biochem. Biophys., 163,
426-428).
The oligoside is condensed with a compound comprising one (or many)
amine function(s) in the presence of sodium cyanoborohydride in
alkaline medium; the imine formed between the reducing sugar and
the amine is reduced by the sodium cyanoborohydride into a
secondary amine.
For example:
The yield of this reaction varies according to the size of the
partners and is usually between 5 and 70%.
There is a destruction of the reducing sugar, which is not
desirable.
French patent number 2 227 823 concerns the N-osides of L
pyrrolidone-2-carboxylic-5 acid, derivatives of said acid and their
procedure of preparation, consisting of condensing the L
pyrrolidone-2-carboxylic-5 acid and/or the L-glutamic acid and/or a
salt of these acids with an ose or an oside, with reducing or
non-reducing properties.
Following the examples, the reducing sugars are all monosaccharides
(ketoses or aldoses), while the non-reducing sugars are, for
example, saccharose; taking into consideration the reaction
conditions, the saccharose becomes hydrolyzed into glucose and
fructose.
It should also be noted that, in the procedure of the said French
patent number 2 227 823, condensation takes place preferably in an
aqueous medium, at a temperature between 50.degree. C. and
150.degree. C., preferably at approximately 105.degree. C.
These operating conditions require working with very concentrated
solutions of sugar and acid which are inapplicable in the
preparation of oligosides; the solubility of oligosides decreases
rapidly while the number of sugars increases.
Furthermore, the condensation reaction between the sugar and the
acid being a thermic dehydration, it is inapplicable to the
preparation of oligosides because of the fragility of the oside
linkages, which are easily hydrolyzed in hot conditions (see for
example the case of saccharose).
Thermic condensation presents, moreover, the drawback of giving
colored products, which are the evidence of degradation reactions
and require a further step of purification.
The procedure thus does not allow obtaining anything but
derivatives of monosaccharides and is not adapted to the production
of derivatives of oligosides.
SUMMARY OF THE INVENTION
One of the aspects of the invention is to propose new derivatives
of glycosylamine in which the oligosides are fixed on at least one
molecule, matrix or particle.
One of the aspects of the invention is to propose derivatives of
glycosylamine, stable in aqueous medium, able to be fixed on at
least one molecule, matrix or particle.
Another object of the present invention is to furnish a process for
the preparation of oligosides fixed on at least one molecule, one
matrix, or one particle, easy to carry out, presenting a reduced
number of steps and allowing to obtain a yield which can, in
practice, reach substantially 100%.
Another aspect of the invention is the ability to fix oligosides
covalently on at least one molecule, matrix, or particle, always
preserving the functional capacity of the oligosides.
The invention relates to new compounds comprising one or several
oligosides, each of said oligosides being fixed covalently on one
or several molecules, matrices or particles, specifically on one,
two or three, via an intermediary molecule possessing a nitrogen
atom carried by an .alpha. carbon of a C.dbd.O group and one or
several functional groups, in particular one, two, or three, the
covalent bond between said intermediary molecule and the oligoside
taking place by the intermediary of the aforementioned nitrogen,
and the covalent bond between said intermediary molecule and the
aforementioned molecule, the aforementioned matrix, the
aforementioned particle, or the aforementioned molecules, the
aforementioned matrices, the aforementioned particles taking place
by the intermediary of the aforementioned functional group or
groups of the said intermediary molecule and the appropriate
functional groups on the molecule(s), matrix(ces), or
particle(s).
The term oligosides corresponds to the presence of at least two,
advantageously to the presence of more than two sugars, and
advantageously to at least 4.
The oligosides entering into the constitution of the compounds of
the invention are such that before the linkage with the
intermediary molecule, they present a terminal reducing sugar.
Because of this, the covalent linkage between the said intermediary
molecule and the oligoside takes place by the intermediary of the
nitrogen atom of the intermediary molecule and of the anomeric
carbon of the terminal reducing sugar, it is to say the carbon in
position 1 of the terminal reducing sugar of the aldose series, or
the carbon in position 2 of the terminal reducing sugar of the
cetose series.
The invention is of compounds of the general formula (I)
##STR1##
in which:
a=0 or 1,
j=0 or 1,
b=0 or 1,
p=2 to 4, in particular 2,
provided that
a=b=0, when j=1, which leads to the presence of a cyclic
molecule,
or a=b=1, when j=0 which implies the absence of the
(CH.sub.2).sub.p group,
D represents a residue of an organic acid of the formula DCO.sub.2
H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in
particular CH.sub.3,
Z represents
B, B being H, an alkyl of 1 to 10 carbon atoms or a side chain of
an a amino acid, natural or synthetic, such as CH(CH.sub.3).sub.2,
CH.sub.2 OH, CH.sub.3, and preferably H, or
B'-P', B' being an alkylene chain of 1 to 10 carbon atoms or a side
chain on an .alpha. amino acid, natural or synthetic, the said
chains containing a group derived from a functional group able to
be activated, such as carboxylic, thiol, hydroxyl, or amine, free
or protected, preferably protected, P' having the significations
indicated hereafter,
X represents: ##STR2##
m being an integer from 0 to 10, preferably from 0 to 5 and
advantageously 1 or 2, k=0 or 1
Q representing OH, OCH.sub.3, OCH.sub.2 --C.sub.6 H.sub.5,
O--C.sub.6 H.sub.5, O--C.sub.6 F.sub.5, ##STR3##
R representing a group possessing an alcohol, phenol, thiol, or
amine function,
P being such as is defined hereafter,
A.sub.i representing an organic radical such as an alkylene chain
of 1 to 10 carbon atoms, in particular (CH.sub.2).sub.n
--W--(CH.sub.2).sub.n', n+n' representing an integer from 0 to 10,
W representing CHY, Y being H, an alkyl from 1 to 6 linear or
branched carbon atoms, an .alpha. amino acid residue, natural or
synthetic, or W representing an aromatic compound, in particular
phenylgroup,
P, P' and P" are identical or different and represent:
a matrice as a support for the affinity chromatography;
a bead of gold, of latex, for histology and cytology;
a protein for the visualization, purification, etc., in particular
1) specific receptors of osides, receptors which are called
lectins, adhesins, agglutinins, etc., or 2) proteins with or
without enzymatic activity, which have an affinity for the osides,
in particular the glycosyltransferases, such as sialyltransferase,
sulfotransferases, phosphotransferases, exoglycosidases, or
endoglycosidases;
a lipid for the characterization of the preceding receptors;
oligonucleotides to selectively increase their capture by target
cells;
a protein or polymers for the targeting of drugs, oligonucleotides
or genes;
P, P' and P" possessing at least one function allowing a
condensation reaction by reaction with an oligopeptide, for
example
an amine function (--NH.sub.2) allowing the formation of an amide
with an active ester, an amidine with an imidate, a thiourea with
an isothiocyanate,
a thiol function (--SH) allowing the formation of a disulfide
bridge with an oligopeptide containing a thiol, a thioether with an
oligopeptide containing a maleimide group, or halogeno-alkyle, or a
halogeno-alkanoyle,
a phenol (--C.sub.6 H.sub.4 OH) allowing the formation of an azoic
with an oligopeptide containing a diazoide,
provided that if Z represents B'--P', and/or X comprises P and/or
P" in the formula.
In formula I and in those which follow, the oligosidyl group
derives from an oligoside whose terminal sugar is reductive.
It is recognized that in the definition of compounds of the general
formula (I) indicated here above, the above mentioned intermediary
molecule includes in its structure the following chemical entity:
##STR4##
in which D, a, b, j and p are such as defined above, Z.sub.1 and
X.sub.1 comprise one or many functional groups able to form at
least one bond with one (or many) molecule(s), or one (or several)
matrix (ces), hereabove mentioned, and Z.sub.1 and X.sub.1 being
more precisely defined in that which follows (see products of
formula Ia defined hereinafter).
In the definitions above, the amino acids implied have
configuration L or D, and preferably L.
The compounds of the invention are thus constituted of one or many
oligosides (identical or possibly different), each of these
oligosides being attached:
to a molecule, matrix or particle, by the intermediary of either a
functional group X, or a functional group Z,
or to two molecules, matrices or particles, by the respective
functional groups X and Z, or by the intermediary of two functional
groups X, or to three molecules, matrices or particles, by the
intermediary of functional groups X and Z.
It should be noted that the general formula (I) represents only the
following possibilities:
a single oligoside fixed to a molecule, matrice, or particle,
P,
a single oligoside fixed to a molecule, matrice, or particle,
P',
a single oligoside fixed to a molecule, matrice, or particle,
P",
a single oligoside fixed to two molecules, matrices, or particles,
P and P' respectively, or P' and P", or P and P',
a single oligoside fixed to three molecules, matrices, or
particles, P, P', and P".
The reason for such representation is to not complicate the
comprehension of the general formula (I). But one can envision the
possibility of many oligosides (identical or possibly different)
being fixed either on a molecule, matrix, or particle, P, or on a
molecule, matrix or particle, P', or on a molecule, matrix or
particle, P", or that many oligosides (identical or possibly
different) could be fixed on two molecules, matrices or particles,
P and P', or P and P", or P' and P", or that many oligosides
(identical or possibly different) could be fixed on three
molecules, matrices or particles, P, P' and P".
As an example of B' one can mention:
##STR5##
or
or
As an example of P, or P' or P", on can mention polylysine, in
particular gluconoylated polylysine.
According to an advantageous mode of embodiment of the invention,
one or many oligosides are linked to the same molecule, matrice or
particle, P, constituting compounds able to be represented by the
general formula (II) ##STR6##
in which:
a=0 or 1,
j=0 or 1,
b=0 or 1,
p=2 to 4, in particular 2,
provided that
a=b=0, when j=1, which leads to the presence of a cyclic
molecule,
or a=b=1, when j=0 which implies the absence of the
(CH.sub.2).sub.p group,
D represents a residue of an organic acid of the formula DCO.sub.2
H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in
particular CH.sub.3,
B represents H, an alkyl of 1 to 10 carbon atoms, or a side chain
of an .alpha. amino acid such as CH(CH.sub.3).sub.2, CH.sub.2 OH,
CH.sub.3, and preferably H,
X represents:
either the group [NH--(A.sub.i)--CO].sub.m --P,
or the group [NH--(A.sub.i)--CO].sub.m --R--P
m being an integer from 0 to 10, preferably from 0 to 5 and
advantageously 1 or 2, R and P being as defined hereinafter,
A.sub.i represents an organic radical such as an alkylene chain of
1 to 10 carbon atoms, in particular (CH.sub.2).sub.n
--W--(CH.sub.2).sub.n ', n+n' representing an integer from 0 to 10,
W representing CHY, Y being H, an alkyl from 1 to 6 linear or
branched carbon atoms, an .alpha. amino acid residue, natural or
synthetic, or W representing an aromatic compound, in particular
phenyl,
R represents a group possessing an alcohol, phenol, thiol, or amine
function,
P represents:
a matrice as a support for affinity chromatography;
a bead of gold, of latex, for histology and cytology;
a protein for the visualization, purification, etc.,
specific receptors of osides, receptors which are called lectins,
adhesins, agglutinins, etc.,
a lipid for the characterization of the preceding receptors;
oligonucleotides to selectively increase their capture by target
cells;
a protein or polymers for the targeting of medicines,
oligonucleotides or genes.
An advantageous class of compounds according to the invention
fulfills the general formula (III) ##STR7##
in which X represents [NH--(A.sub.i)--CO].sub.m --P or
[NH--(A.sub.i)--CO].sub.m --R--P, A.sub.i, m, P and R having the
significations indicated above.
An advantageous class of compounds according to the invention
fulfills the formula (III): ##STR8##
in which X represents: ##STR9##
A.sub.i, m, P, R and P" having the significations indicated
above.
Another advantageous class of compounds according to the invention
has as a general formula (IV): ##STR10##
in which D and B have the significations indicated above, and X
represents [NH--(A.sub.i)--CO].sub.m --P or
[NH--(A.sub.i)--CO].sub.m --R--P, A.sub.i, m, R and P having the
significations indicated above.
Another advantageous class of compounds according to the invention
fulfills the formula (IV): ##STR11##
in which D and B have the significations indicated above, and X
represents: ##STR12##
A.sub.i, m, R, P and P" having the significations indicated
above.
Another advantageous class of compounds according to the invention
has as a general formula (V): ##STR13##
in which X represents [NH--(A.sub.i)--CO].sub.m --P or
[NH--(A.sub.i)--CO].sub.m --R--P, P, A.sub.i, m and R having the
significations indicated above.
The invention also relates to new products, able in particular to
serve as intermediary products for the preparation of compounds of
the invention, said products comprising an oligoside linked to a
molecule possessing a nitrogen atom, carried by an a carbon of
.alpha. C.dbd.O group, and at least one functional group, in
particular one, two or three functional groups, the covalent link
between the oligoside and the molecule being made by the
intermediary of the said nitrogen atom.
The products of the invention advantageously fulfill the general
formula (Ia): ##STR14##
in which:
a=0 or 1,
j=0 or 1,
b=0 or 1,
p=2 to 4, in particular 2,
provided that
a=b=0, when j=1, which leads to the presence of a cyclic
molecule,
or a=b=1, when j=0 which implies the absence of the
(CH.sub.2).sub.p group,
D represents a residue of an organic acid of the formula DCO.sub.2
H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in
particular CH.sub.3,
Z.sub.1 represents
B, B being chosen from among: H, an alkyl chain of 1 to 10 carbon
atoms, or a lateral chain of an .alpha. amino acid such as
CH(CH.sub.3).sub.2, CH.sub.2 OH, CH.sub.3, and preferably H, or
B', B' being chosen from among: an alkylene chain of 1 to 10 carbon
atoms, or a lateral chain of an .alpha. amino acid, said chains
containing a functional group such as carboxylic, SH, OH or amine,
free or protected,
X.sub.1 represents
the group [NH--(A.sub.i)--CO].sub.m --R,
or [NH--(A.sub.i)--CO].sub.m --Q
R representing a group possessing an alcohol, phenol, thiol, or
amine function,
Q representing OH, OCH.sub.3, OCH.sub.2 --C.sub.6 H.sub.5,
O--C.sub.6 H.sub.5, O--C.sub.6 F.sub.5, ##STR15##
m being an integer from 0 to 10, preferably from 0 to 5 and
advantageously 1 or 2,
A.sub.1 represents an organic radical such as an alkylene chain
from 1 to 10 carbon atoms, in particular (CH.sub.2).sub.n
--W--(CH.sub.2).sub.n', n+n' representing an integer from 1 to 10,
W representing CHY, Y being H, an alkyl from 1 to 6 linear or
branched carbon atoms, an .alpha. amino acid residue, natural or
synthetic, or W representing an aromatic compound, in particular
phenyl.
These groups are derivatives of acylated glycosylamine.
The derivatives of acylated glycosylamine are stable, in an aqueous
medium in a large range of pH on each side of neutrality. This
signifies that a hydrolysis of less than 1% at pH 5-8 is produced,
over 24 h, whatever the temperature may be between 0 and 95.degree.
C.
The said products of formula (Ia) are able to be utilized as
such.
The said products of formula (Ia) are also able to be utilized in
order to prepare the compounds of the invention of formula I.
An advantageous class of products according to the invention
fulfills the general formula (IIa): ##STR16##
in which:
a=0 or 1,
j=0 or 1,
b=0 or 1,
p=2 to 4, in particular 2,
provided that
a=b=0, when j=1, which leads to the presence of a cyclic
molecule,
or a=b=1, when j=0 which implies the absence of the
(CH.sub.2).sub.p group,
D represents a residue of an organic acid of the formula DCO.sub.2
H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in
particular CH.sub.3,
B represents H, an alkyl chain of 1 to 10 carbon atoms, or a side
chain of an .alpha. amino acid such as CH(CH.sub.3).sub.2, CH.sub.2
OH, CH.sub.3, and preferably H,
m being an integer from 1 to 10, preferably from 0 to 5 and
advantageously 1 or 2,
A.sub.1 represents an organic radical such as an alkylene chain
from 1 to 10 carbon atoms, in particular (CH.sub.2).sub.n
--W--(CH.sub.2).sub.n', n+n' representing an integer from 1 to 10,
W representing CHY, Y being H, an alkyl from 1 to 6 linear or
ramified carbon atoms, an .alpha. amino acid residue, natural or
synthetic, or W representing an aromatic compound, in particular
phenyl,
R represents a compound possessing an alcohol, phenol, thiol or
amine function.
The said products of formula (IIa) are able to be used to prepare
the compounds of formula (II).
Another advantageous class of products according to the invention
fulfills the general formula (IIIa): ##STR17##
in which A.sub.1, m and R have the significations indicated
above.
The said products of formula (IIIa) are able to be utilized to
prepare the compounds of formula (III).
Another advantageously class of products according to the invention
fulfills the general formula (IVa): ##STR18##
in which D, B, m, A.sub.1 and R have the significations indicated
above.
The said products of formula (IVa) are able to be used to prepare
the compounds of formula (IV).
Another advantageously class of products according to the invention
fulfills the general formula (Va): ##STR19##
in which A.sub.1, m and R have the significations indicated
above.
The said products of formula (Va) are able to be used to prepare
the compounds of formula (V).
The product of formula (VI): ##STR20##
in which B, A.sub.1, m and R have the significations indicated
above, are intermediary products obtained during the preparation of
the products defined above and are new.
The products of formula (VII) ##STR21##
in which p, A.sub.1, R and m have the significations indicated
above, are intermediary products obtained during the preparation of
the products defined above and are new.
In all of the compounds or products of the invention, R can
represent the following radicals: ##STR22##
--NH-pC.sub.6 H.sub.4 --N.dbd.C.dbd.S and its precursors:
--NH-pC.sub.6 H.sub.4 --NO.sub.2
--NH-pC.sub.6 H.sub.4 --NH.sub.2
--NH--CH.sub.2 --(CH.sub.2).sub.m
--C(.dbd.NH.sub.2.sup.+)OCH.sub.3
--NH--CH.sub.2 --(CH.sub.2).sub.m --CN
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--CH.sub.2
--(CH.sub.2).sub.m --C(.dbd.NH.sub.2.sup.+)OCH.sub.3
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--CH.sub.2
--(CH.sub.2).sub.m --CN
these compounds allowing for the addition on one of the amino
compounds.
R can also represent ##STR23##
derivative of maleimide ##STR24##
derivative of m-maleimidyl benzoyl ##STR25##
derivative of N-methylmaleimidyl p-cyclohexylcarboxy-
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--CH.sub.2
-T
T=Br, I, Cl
derivative of halogenoacetyl
--NH--CH.sub.2 --CH.sub.2 --NH--CO--CH.sub.2 --(CH.sub.2).sub.m
--S--S-Pyr
derivative of dithiopyridine
N.B.: -Pyr: -2-pyridine
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --S--S-Pyr
derivative of dithiopyridine
these compounds allowing the addition on a thiol.
R can also represent: ##STR26##
derivative of biotin
this compound including a biotin residue which allows the formation
of a complex with avidine or streptavidine.
R can also represent: ##STR27##
derivative of 4-azidobenzoyl ##STR28##
derivative of 4-azido 2-nitrobenzoyl ##STR29##
derivative of 4-azido 2-nitroanilide ##STR30##
derivative of 7-azido 4-methylcoumarine 3-acetyl ##STR31##
derivative of 4-azidosalicyclique or of 4-azido
2-hydroxybenzoyle
these compounds allowing the covalent fixation on a receptor, an
enzyme, an antibody, specific to the glucidic part by photonic
activation.
R can also represent:
--NH--(CH.sub.2).sub.m -pC.sub.6 H.sub.4 OH
--NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2
--NH--CO--(CH.sub.2).sub.m -pC.sub.6 H.sub.4 OH
these compounds allowing the fixation of one or two iodine atoms,
specifically of a radioactive iodine atom.
R can also represent: ##STR32##
derivative of fluoresceine ##STR33##
derivative of dansyl ##STR34##
derivative of 7-amino 4-methylcoumarin 3-acetyl ##STR35##
derivative of amino-fluoresceine ##STR36##
derivative of nitrobenzoxadiazole
these fluorescent compounds allowing visualizing the position of
the oligosidylpeptide in a cell, a tissue, an organ, on a gel or an
electrophoresis band, etc.
In the compounds of the invention, P can represent an oligopeptide,
or a polypeptide, in particular gluconoylated polylysine, and P' or
P" can represent an oligonucleotide, or R can represent
fluoresceine or one of its derivatives or another fluorescent
derivative, and P' or P" can represent an oligonucleotide. P can
also represent a therapeutic agent or any molecule of interest.
In the compounds and products of the invention, the oligosidic
residue comprises from 2 to 50 oses and in particular is chosen
from
lacto-N-tetraose Gal.beta. 3 GlcNAc.beta. 3 Gal.beta. 4 Glc
neolacto-N-tetraose Gal.beta. 4 GlcNAc.beta. 3 Gal.beta. 4 Glc
Group H Fuc .alpha. 2 Gal.beta. 3 Gal.beta. 4 Glc Lewis.sup.a
Gal.beta. 3 GlcNAc.beta. 3 Gal.beta. 4 Glc Fuc .alpha. 4-.uparw.
Lewis.sup.x Gal.beta. 4 GlcNAc.beta. 3 Gal.beta. 4 Glc Fuc .alpha.
3-.uparw. Lewis.sup.b Fuc .alpha. 2 Gal.beta. 3 GlcNAc .beta. 3
Gal.beta. 4 Glc Fuc .alpha. 4-.uparw. Lewis.sup.y Fuc .alpha. 2
Gal.beta. 4 GlcNAc.beta. 3 Gal.beta. 4 Glc Fuc .alpha. 3-.uparw.
Disialolacto-N-tetraose Neu 5Ac.alpha. 3 Gal .beta. 3 GlcNAc .beta.
3 Gal .beta. 4 Glc Neu 5Ac.alpha. 6-.uparw. Three antenna complex
type ##STR37## Silaylactose 3 Neu 5Ac .alpha. 3 Gal 4 Glc
Sialylactose 6 Neu 5Ac .alpha. 6 Gal .beta. 4 Glc Disialylactose 3
Neu 5Ac .alpha. 8 Neu 5Ac .alpha. 3 Gal .beta. 4 Glc
simple or complex osides recognized by the lectin membranes, and
chosen from:
a. Asialo-oligoside of type lactosamine triantenna: receptor of
asialoglycoprotein ##STR38##
b. Asialo-oligoside of type lactosamine tetraantenna: receptor of
asialoglycoprotein ##STR39##
c. Lewis.sup.x : LECAM 2/3 ##STR40##
d. Sialyl Lewis.sup.x : LECAM 3/2 ##STR41##
e. Derivative of Lewis.sup.x sulfate (HNK1): LECAM 1 ##STR42##
f. Oligomannoside: receptor of mannose ##STR43##
g. Phosphorylated oligomannoside: receptor of mannose 6 phosphate
##STR44##
h. Oligosaccharide of sulfated lactosamine type: receptor of
sulfated GalNAc 4 ##STR45##
To prepare the compounds of the invention, it is necessary to first
prepare the products (derivatives of acylated glycosylamine), which
serve in particular as intermediaries for the preparation of said
compounds of the invention.
The invention also concerns a procedure of preparation of products
(derivatives of acylated glycosylamine) defined above,
characterized in that:
one condenses an oligoside having a free terminal reducing sugar,
on the nitrogen atom of an intermediary molecule, this nitrogen
atom belonging to the amine group, linked to a carbon atom placed
in .alpha. of a C.dbd.O group, the intermediary molecule possibly
possessing a lateral chain containing a functional group such as
OH, SH, NH.sub.2 or COOH, free or protected, this intermediary
molecule being chosen from the following intermediary molecules: a
amino acid, natural or synthetic, derivative of .alpha. amino acid,
amino acid in N-terminal position of a peptide, or a peptidic
derivative, possibly in presence of a catalyst such as imidazole,
in a solvent appropriate for obtaining a derivative of
glycosylamine in which the terminal ose of the oligoside conserves
its cyclic structure, and in which the semiacetalic hydroxyl is
replaced by the .alpha. amine of one of the said starting
molecules,
while the intermediary molecule does not possess a lateral chain
containing a functional group such as described above, or a side
chain in which the functional group is possibly protected, one
acylates the derivative of glycosylamine obtained at the issue of
the preceding step by the addition of an organic acid activated by
a classical activator such as carbonyl diimidazole, BOP
(benzotriazolyl N-oxy-tris(dimethylamino) phosphonium
hexafluorophosphate) or HBTU
(O-benzotriazol-1-yl-N,N,N',N',tetramethyluronium
hexafluorophosphate) to obtain a derivative of N-acylated
glycosylamine, followed possibly by a deprotection of the
functional group of the said lateral chain, in view of a possible
substitution,
while the intermediary molecule possesses a side chain containing a
carboxylic group, one activates the said carboxylic group to induce
an intramolecularly reaction with the said .alpha. amine, leading
to a cyclization inside the said intermediary molecule, to obtain a
derivative of N-acylated glycosylamine.
while the intermediary molecule possesses a side chain containing a
carboxylic group, one can add the acylation agent in an active
ester form.
The said procedure of preparation of the invention of derivatives
of glycosylamine thus includes two steps, one step of condensation
of an oligoside on an intermediary molecule to obtain a derivative
of glycosylamine, and another step of acylation of said derivative
of glycosylamine.
In the step of acylation under consideration in the procedure of
the invention, one always uses an activator, in case the
intermediary possesses or not a side chain containing a functional
group.
Furthermore, it should be specified that the condensation step of
the oligoside on the intermediary molecule, to obtain a
glycosylamine derivative, as well as the step of acylation of said
derivative of glycosylamine are done in presence of appropriate
organic solvents.
One of the advantages of using an organic solvent is in particular
to allow coupling to the oligoside peptides and derivatives which
are slightly or very slightly soluble in water.
These cases can be schematized in the following manner:
1) The intermediary molecule does not possess a side chain
containing a functional group, ##STR46##
R.sub.1 representing a residue of an organic molecule without a
protected functional group, or R.sub.1 being also able to represent
H;
R.sub.2 representing a residue of an organic molecule such as
--CO--R2, either an ester or an amide;
R.sub.3 representing a residue of an organic molecule preferably
not comprising a free functional group.
The group R.sub.3 --CO.sub.2 H+activator can be replaced by the
activated product or by an anhydride.
In that which precedes, one can also include the case where R.sub.3
possesses a functional group, and in this case, one should refer to
paragraph 1a here below.
1a) the intermediary molecule does not possess the side chain
containing the functional group, but the agent of acylation is
bifunctional, ##STR47##
R.sub.3 representing a residue of an organic molecule comprising a
second functional group such as, in particular SH, free or
protected, or --CO.sub.2 H.
Alternatively, the group R.sub.3 --CO.sub.2 H+activator can be
replaced by the product of activation: R.sub.3 --CO-activated.
For example, ##STR48##
or a cyclic anhyride ##STR49##
for example with integer n equal to 1, 2, 3 or 4; or a thioester:
##STR50##
with whole number n equal to 1, 2, 3 or 4, preferably n=2.
While the acylation of the nitrogen linked to the oligoside is
acylated by a cyclic anhydride, the product obtained is of the
type:
The carboxylic group is able to be used for a coupling reaction on
an organic molecule or a matrix, or a particle possessing a
functional group (hydroxyl or amine for example).
While the acylation of the nitrogen linked to the oligoside is
acylated by a cyclic thio ester, the product obtained is of the
type:
The thiol group is able to be used for a coupling reaction on a
soluble or insoluble molecule, able to be substituted by a thiol,
for example a dithiopyridine or a maleimide derivative.
2) The intermediary molecule possesses a side functional chain and
cyclization is not carried out, ##STR51##
R.sub.2 and R.sub.3 having the significations indicated above, and
R.sub.4 representing a residue of an organic molecule possessing a
functional group, specifically a carboxylic group, R.sub.4
representing specifically CH.sub.2 --CH.sub.2 --CO.sub.2 H.
In this case, one uses preferentially a product of activation of
R.sub.3 --CO.sub.2 H such as is defined above.
The functional group contained in R.sub.4 is available for a
condensation reaction or substitution on a soluble or insoluble
molecule on a matrice, or a particle comprising of a group able to
give a covalent link with the functional group of R.sub.4, for
example an amine while R.sub.4 comprises of a carboxylic group.
3) The intermediary molecule possesses a side chain containing a
functional carboxylic group, and one effects a cyclization, and one
can fix the molecule on 1 or 2 molecule(s), matrix(ces) or
particle(s). ##STR52##
R.sub.2 having the significations indicated above,
R.sub.5 --CO.sub.2 H being a residue of an organic molecule such as
--(CH.sub.2).sub.n --CO.sub.2 H
n being an integer from 1 to 10, preferably 2 or 3.
The glycopeptide thus obtained can be used to react with an
existing functional group in a free state or transformed into an
active group on R.sub.2. For example, if R.sub.2 represents the
para-nitroanilide group, the NO.sub.2 group is reduced to NH.sub.2,
then transformed into isothiocyanate --N.dbd.C.dbd.S, which is an
excellent reactor with alcohols and to amines.
As examples of intermediary molecules, one can cite: ##STR53##
with n=1 to 10.
The invention also concerns the preparation of compounds of the
invention, characterized in that one makes a reaction with one or
many products (derivatives of glycosylamine acylates) of the
invention, carrying either an R group such as defined above,
activated or able to be activated, or an A.sub.i group, containing
a functional group able to react on a molecule, matrix or particle,
P, P' or P" respectively, containing a functional group, to obtain
a product of type:
The invention also concerns the preparation of compounds of the
invention, characterized in that one can react one or many products
(derivatives of acylated glycosylamine) of the invention, carrying
in one part an R group such as defined above, activated or able to
be activated, and in another part a B' group containing an
activated or able to be activated group, or an A.sub.i group
containing a functional on molecules, matrices or particles P and
P', or molecules, matrices or particles P and P".
The invention also concerns the preparation of compounds of the
invention, characterized in that one can react one or many products
(derivatives of acylated glycosylamine) of the invention, carrying
in one part an R group such as is defined above, activated or able
to be activated, in another part a B' group containing an activated
or able to be activated group, and an A.sub.i group containing a
functional group respectively on molecules, matrices or particles
P, B' and P'.
The molecules, matrices or particles entering into the preparation
of compounds of the invention are able advantageously to be a
natural or synthetic molecule, soluble or not in an organic,
aqueous or hydroorganic solvent, a nano particle, a lipid vesicle,
a matrix insoluble in an organic aqueous or hydroorganic solvent, a
latex bead or a gold bead, etc.
In detail, concerning a procedure according to the invention, the
oligoside, having a free reducing sugar, is placed in appropriate
solvent, dimethylsulfoxyde, N-methylpyrrolidone or
dimethylformamide, for example, in presence of an equal quantity or
two equal quantities of a starting molecule chosen from: a natural
or synthetic amino acid, a peptide, an amino acid derivative or a
peptide derivative.
By appropriate solvent, one designates a solvent allowing both, the
solubilisation of compounds to be condensed, and the solubilisation
of compounds resulting from the condensation.
The principal product of the reaction is the product of
condensation of the type "derivative of glycosylamine": the
terminal reducing sugar conserves its cyclic structure, its
semiacetalic hydroxyl is replaced by the .alpha. amine of the amino
acid, by the derivative of the amino acid, or by the amino acid in
N terminal position of a peptide or a peptide derivative.
In a second step, the derivative of glycosylamine thus formed is
acylated by the addition of an organic activated acid or in the
case of a amino acid carrying a lateral chain containing a
functional group such as a lateral carboxylic chain, as in the case
with glutamic acid or its homologues, by addition of an activator
of the carboxylic group.
The product thus formed is a derivative of N-acylated
glycosylamine.
The acylated glycosylamine derivatives are isolated by gel
filtration chromatography or by any other classic purification
technique known in the field.
The derivatives of acylated glycosylamine are then used to
substitute a compound (protein, lipid, nucleic acid,
oligonucleotide, polylysine, insoluble polymers, latex beads, gold
beads, etc.).
One takes advantage of the amino acid fraction, or of the peptide
or of a peptide substitute in order to effect the condensation
reaction, so that the oligoside conserves all of its properties and
its accessibility to serve as substrates or as recognition
signals.
According to the following general scheme: ##STR54##
For example, the condensation of lactose with
glycylparanitroanilide is expressed ##STR55##
The addition of acetic acid and an organic acid activator leads to:
##STR56##
In the example chosen, the nitro group can be further reduced
quantitatively into amine, then the amine is transformed
quantitatively into isothiocyanate (according to Roche et al. 1983,
J. Cell Biochem. 22, 131-140, Monsigny et al. 1984, Biol. Cell 21,
187-196).
The compound thus activated can react in a slightly alkaline medium
with an amine carried by a protein, lipid, polymer, (polylysine for
example), a solid support comprising amine groups, or an
oligonucleotide substituted by an amine, or with an amine carried
by a molecule or an appropriate body, such as NH.sub.2 --P.
One can write, as an example, the following reaction scheme:
##STR57##
In the case where the amino acid or the derivative is an amino acid
possessing a side chain containing a carboxylic group as is the
case for glutamate, the reaction is expressed, for example:
##STR58##
The addition of an activator of carboxylic group leads to the
following expected product: ##STR59##
In an analogous way, this compound could be activated and could
react on an amine NH.sub.2 --P, to give the final product:
##STR60##
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents the profile of elution of the
.beta.-lactosyl-pyroGlu-pNA obtained by high performance anion
exchange chromatography as described in Example 11 below.
FIG. 2 represents the profile elution of the Lewis.sup.A
/Lewis.sup.X -pyroGlu-pNA obtained by high performance anion
exchange chromatography as described in Example 12 below.
EXAMPLES
Example 1
Preparation of a derivative of acylated glycosylamine: N-acetyl
lactosyl .beta.-glycyl-pNA
The amido paranitrophenyl (0.1 mmole) and the lactose (0.1 mmole)
are dissolved in 1 ml of dimethylsulfoxyde.
The solution is maintained at 50.degree. C. for 48 h. 0.1 mmole of
Gly-pNA in 0.5 ml of dimethylsulfoxyde is added at 12 h, 24 h and
36 h. The solution is cooled to 25.degree. C.
Next, 0.44 mmol of BOP, hexafluorophosphate, benzotriazolyl 1
yl-tris (dimethylamino) phosphonium, and 0.44 mmole of
diisopropylethylamine acetate is added and the solution is agitated
for 3 h at 25.degree. C.
The desired product is purified by molecular sieving on a Ultrogel
GF05 column (90.times.2.3 cm) using 0.1 M acetic acid as a solvent,
containing 3% of n-butanol; before the injection, the products of
the reaction are diluted by addition of 7.5 ml of solvent of
chromatography.
The desired product is eluted first, followed by excess of reactive
agents and by dimethylsulfoxyde.
The product: N-acetyl lactosyl.beta.-Gly-pNA is obtained by
lyophilisation of the solution eluted from the column.
Example 2
Preparation of a derivative of intramolecularly acylated
glycosylamine: N-lactosyl.beta.-pyroGlu-pNA
.alpha. glutamyl paranitroanilide (Glu-pNA) (0.1 mmole) and lactose
(0.1 mmole) are dissolved in 1 ml dimethylsulfoxyde. The solution
is maintained at 50.degree. C. for 48 h. 0.1 mmole of Glu-pNA
dissolved in 0.5 ml of dimethylsulfoxyde is added at 12 h, 24 h,
and 36 h. The solution is cooled to 25.degree. C.
0.44 mmole of BOP is then added and the solution is stirred for 3 h
at 25.degree. C.
The desired product is purified under the same conditions as in the
preceding example.
Example 3
Preparation of an organic compound, the side chain of which
contains a functional group and use of the functional group to form
a conjugate with an oligonucleotide
The oligoside is incubated in presence of two to four equivalents
of the derivative S-(thio-2-pyridine)cysteinyl-p-nitroanilide
##STR61##
in solution in N-methylpyrrolidone (or in dimethylsulfoxyde or
N-dimethylformamide) and in presence of four equivalents of
imidazole for 20 h at 50.degree. C. The solution is cooled to
25.degree. C.
Ten equivalents of acetic acid, imidazole, and BOP are then added.
The acylation reaction takes place in a half hour.
The glycopeptide is isolated by size exclusion chromatography
(column of Ultrogel GF05, for example), in 0.1M acetic acid. The
fraction containing the purified glycopeptide is frozen and
lyophilized.
The glycopeptide dissolved in 0.1M sodium acetate buffer, pH6, is
reduced by addition of an equivalent of TCEP
(tris-carboxyethylphosphine) at 25.degree. C. for 30 min (see K.
Arar et al.; 1993, Tetrahedron Letters 34, 8087-8090, J. A. Burns
et al., 1991, J. Org. Chem., 56, 2648-2650). Added next is an
equivalent of an oligonucleotide substituted on its 5' extremity by
a substitute terminated by a dithio-2-pyridine group.
The glycopeptide-oligonucleotide conjugate formed has the following
general structure: ##STR62##
in which X represents NH-p-C.sub.6 --H.sub.4 --NO.sub.2, and 5'
represents the arm bridging the first S dithio-2-pyridine to the
primary hydroxyl of the first nucleotide of the
oligonucleotide.
Example 4
Preparation of lactosyl-pyroglutamyl-para-nitroanilide (in
dimethylsulfoxyde)
The lactose Gal.beta.4Glc (0.15 mmole) is dissolved in 1.25 ml
dimethylsulfoxyde (CH.sub.3 --SO--CH.sub.3). 0.30 mmole of Glu-pNA
in 1.25 ml dimethylsulfoxyde containing 0.6 mmole imidazole
(pNA=para nitro-anilide) is added. The solution is kept at
50.degree. C. for 20 h, then cooled to 25.degree. C. More than 95%
of the lactose is transformed into glycopeptide:
lactosyl-Glu-pNA.
0.33 mmole of BOP and 0.6 mmole of imidazole is added. The solution
is stirred for 30 min at 25.degree. C. More than 95% of the
glycopeptide is cyclized into
lactosyl-pyroglutamyl-paranitro-anilide:
pGlu is: the residue pyroglutamyl ##STR63##
Example 5
Preparation of lactosyl-pyroglutamyl-p-nitroanilide (in
N-methylpyrrolidone)
The lactose Gal.beta.4Glc (0.15 mmole) is dissolved in 1.25 ml
N-methylpyrrolidone of the formula: ##STR64##
0.3 mmole of Glu-pNA in 1.25 mmole of N-methyl-pyrrolidone
containing 0.6 mmole of imidazole is added. The solution is kept at
50.degree. C. for 20 h then cooled to 25.degree. C. More than 95%
of the lactose is transformed into glycopeptide: lactosyl-Glu-pNA.
0.33 mmole of BOP and 0.6 mmole of imidazole are added. The
solution is stirred for 30 min at 25.degree. C. More than 95% of
the glycopeptide is cyclized into
lactosyl-pyroglutamyl-p-nitro-anilide.
The analyses are done by high pressure liquid chromatography on a
column on a Dionex apparatus, equipped with an amperometric
detector. The results are calculated in relation to an internal
control (sorbitol) added to the initial lactose solution.
The time of retention of the compounds under standard conditions,
expressed in minutes, are:
Lactose 9.9 .+-. 0.1 Lactosyl-Glu-pNa 21.4 .+-. 0.1
Lactosyl-p-Glu-p-NA 16.2 .+-. 0.1 Imidazole 4.3 .+-. 0.1 Sorbitol
2.7 .+-. 0.1
Example 6
Preparation of a compound of the formula ##STR65##
The preparation may be done according to the following reaction
schema.
The original product indicated hereafter, can be obtained as
previously indicated, in relation to the preparation of the
products of the invention. ##STR66## ##STR67##
The example chosen corresponds to the preparation of a derivative
of oligonucleotide, so that this derivative presents a biological
activity in relation to the sequence of the oligonucleotide chosen
(the oligonucleotide sense, anti-sense, antigen, bait, etc., is
specific to a cellular or viral element of nucleic acid or protein
nature), the oligoside allows the derivative to be selectively
recognized by certain cells which possess a membrane receptor
(lectin) having an affinity for the chosen oligoside. The peptide
allows the derivative--once inside the endosomes (intracellular
vesicles), thanks to the mechanism of endocytosis due to the
membrane lectin--to penetrate the cytosol and then the nuclear
compartments.
The oligonucleotide is an oligomer containing between 10 to 40
nucleotides, preferably 20 to 25. The oligopeptide is an oligomer
comprising between 20 to 40 amino acids, preferably 20 to 25. This
type of derivatives corresponds to a line of compounds able to be
utilized as drugs.
Example 7
Preparation of a compound of formula ##STR68##
The preparation of this compound can be made following the
following reaction schema: ##STR69##
The example chosen corresponds to the preparation of a derivative
of glycopeptidic fluorescent of general utilisation.
The residue of fluoresceine allows for an utilisation of
glycopeptides for the purposes of localisation, visualisation, in a
general manner, for the purposes of analysis, specifically in
fluorescence microscopy.
The glycopeptidic fluorescent derivative linked to a
oligonucleotide can also be used as an antiviral or anticancerous
agent, in order to allow at the same time the study of the
biological activity of the derivative and its intracellular
activity as well as its pharmacokinetic activity in the animal.
In this example, the disulfure bridge is present in the original
compound on an Ai residue of the general formula.
Example 8
Preparation of a compound of formula ##STR70##
What has been said regarding the glycopeptidic derivative of
example 7 also applies to this example. It should be noted that in
example 8 herein considered, the disulfure bridge is present in the
original compound on the Z chain of the general formula.
Example 9
Preparation of glycosylated derivatives of gluconoylated
polylysine
The gluconoylated polylysine is used to transfer genes into animal
cells. Substitution of the gluconoylated polylysine by one or many
glycopeptides allows to make selective the transfer of genes.
The glycosylated derivatives of the gluconoylated polylysine
penetrate preferably (100 to 1000 times more) in cells which
express on their surface a lectin (receptor of oligosides), which
specifically recognizes the oligoside of the glycopeptide linked to
the gluconoylated polylysine.
a) Link of a glycopeptide to the gluconoylated polylysine via a
disulfide bridge.
The gluconoylated polylysine (degree of polymerisation 190;
containing 60 gluconoyle residues), is substituted by a derivative
of the dithiopyridine; the polymer (20 mg; 0.33 .mu.mol) is
dissolved in 0.5 ml of dimethylsulfoxyde.
1 .mu.mol (312 .mu.g) of N-succinimidyl
3-(2-pyridyldithio)propionate and 20 .mu.mol (3.6 .mu.l) of
diisopropylethylamine are added. The solution is stirred at
20.degree. C. for 15 h. The polymer is precipitated by addition of
10 volumes of isopropanol; the precipitate is recovered after
centrifugation (1 800 g, 15 min).
After washing with isopropanol, the polymer is dissolved in a
sodium phosphate buffer, 0.1 M at pH 7.2 (1 ml).
The glycopeptide: oligosylpyroglutamyl amido
ethyldithiopyridine:
Gal.beta.4Glc.beta.-pyroglutamyl-NH--(CH.sub.2).sub.2
--S--S-pyridine (1 .mu.mol) is treated with 1 .mu.mole of TCEP
[(triscarboxyethylphosphine: P(CH.sub.2 --CH.sub.2
--CO.sub.2.sup.-).sub.3)] in a sodium phosphate buffer, 0.1 M (1
ml), for 1 h at 20.degree. C. This solution is added to the
solution of gluconoylated polylysine substituted with
pyridyldithiopropionate. After 1 h at 20.degree. C., the polymer is
precipitated by addition of 10 volumes of isopropanol. The
precipitate is recovered after centrifugation (1 800 g, 15 min) and
washed with isopropanol, then dissolved in water and
lyophilized.
The yield of the coupling reaction under the used conditions is
equal or superior to 90%.
The reactions which are used in this preparation are derived of the
ones described in Midoux, P., Mendes, C., Legrand, A., Rammond, J.,
Mayer, R., Monsigny, M. and Roche, A. C., 1993: Specific gene
transfer mediated by lactosylated poly-1-lysine into hepatoma
cells. Nucleic Acid Research, 21: 871-878, and in Arar, K.,
Monsigny, M., and Mayer, R., 1993: Synthesis of oligonucleotide
peptide conjugates containing a KDEL signal sequence. Tetrahedron
Letters, 34: 8087-8090.
b) Link of a glycopeptide to the gluconoylated polylysine via a
thiourea link.
The gluconoylated polylysine (degree of polymerisation 190;
containing 60 residues of gluconoyle), is substituted by an
activated glycopeptide in the form of phenylisothiocyanate.
The glycopeptide oligosylpyroglutamyl p-nitroanilide is reduced to
a p-amino anilide derivative which is then activated into a
p-cyanato-anilide derivative:
Gal.beta.4Glc.beta.-pyroglutamyl-NH-p-C.sub.6 H.sub.4 --NCS
following a protocol adapted to that described in Roche, A. C.,
Barzilay, M., Midoux, P., Junqua, S., Sharon, N. and Monsigny, M.
(1983): Sugar specific endocytosis of glycoproteins by Lewis lung
carcinoma cells., J. Cell. Biochem., 22: 131-140.
The cyanato-anilide derivative (1 .mu.mole) is dissolved in
dimethylsulfoxyde (1 ml) containing 1 .mu.mole of gluconoylated
polylysine and 4 .mu.moles of diisopropyl ethylamine. The solution
is stirred at 20.degree. C. for 24 h. The glycosylated polymer is
precipitated by addition of 10 volumes of isopropanol; the
precipitate is recovered after centrifugation, washed with
isopropanol, and finally dissolved in water and lyophilized.
In the conditions described, the coupling yield of the glycopeptide
to the gluconoylated polylysine is better than to 95%
Example 10
Preparation of a glycopeptide (oligosylpyroglutamyl-p-nitroanilide)
in dimethylformamide as solvent
The .alpha. glutamyl-p-nitroanilide (0.2 mmoles) and the lactose
(0.1 mmole) are dissolved in 1 ml of dimethylformamide, in presence
of 0.2 mmoles of imidazole. The solution is kept at 50.degree. C.
for 8 h.
The solution is cooled to 25.degree. C., and 0.2 mmole of BOP and
0.2 mmole of imidazole are added and left for 30 min. Under these
conditions, more than 95% of the original oside is transformed into
glycopeptidic derivative. The purification is identical to that
described in using the other solvents.
Example 11
The purity of the prepared product at example 2
(N-lactosyl.beta.-pyroGlu-pNA, which is hereafter designated also
as .beta.-lactosyl-pyroGlu-pNA) was assessed by high performance
anion exchange chromatography and by amperometric detection
(HPAE-PAD) on an apparatus of trademark Dionex.
Concerning this technique, one proceeds as follows:
The osides are ionized in alkaline medium (0.1M sodium hydroxyde)
in the form of polycoolates. Their separation on a cationic resin
(immobilized ammonium ions ) is very efficient. The detection of
the osides is advantageously effected by an amperometric measure in
pulse current. The apparatus used (Dionex) was specially built to
carry out the chromatography in alkaline medium and the
amperometric detection on line.
The retention times (t.sub.r) of the different products were
characterized (see FIG. 1):
Pic t.sub.r (min) Compound 1 4,4 Imidazole 2 10,0 Lactose 3 27,5
.beta.-lactosyl-Glu-pNA 4 22,9 .beta.-lactosyl-pyroGlu-pNA
In FIG. 1:
the first curve (from the top of the sheet) corresponds to the
injection of only lactose,
the second curve corresponds to the injection of the reaction
mixture after 12 h,
the third curve corresponds to the injection of the reaction
mixture after 12 hours+30 minutes of cyclisation.
Example 12
Preparation of a derivative of intramolecularly acylated
glycosylamine: Lewis.sup.A /Lewis.sup.X -pyroGlu-pNA
Lewis.sup.A /Lewis.sup.X (0.06 mmole) is dissolved in 1 ml of
dimethylformamide. 0.12 mmole of Glu-pNA then 0.24 mmole of
imidazole are added. The solution is kept at 50.degree. C. for 15
h. The stabilisation of the glycopeptide is obtained by adding,
after reaction medium brought to 20.degree. C., 0.13 mmole of BOP
and 0.24 mmole of imidazole. After 30 min, the glycopeptide is
cyclized into Lewis.sup.A /Lewis.sup.X -pyroGlu-pNA. The reaction
is followed by HPAE-PAD.
Synthesis of Lewis.sup.A /Lewis.sup.X -pyroGlu-pNA. Profiles of
Dionex elution. The retention times (t.sub.r) of different products
where characterized (see FIG. 2):
Pic t.sub.r (min) Compound 1 4.5 Imidazole 2 8.9/9.4 Lewis.sup.A
/Lewis.sup.X 3 22.5/22.7 Lewis.sup.A /Lewis.sup.X -Glu-pNA 4
17.4/17.6 Lewis.sup.A /Lewis.sup.X -pyroGlu-pNA
In FIG. 2:
the first curve (from the top of the sheet) corresponds to the
injection of the reaction mixture after 15 minutes,
the second curve corresponds to the injection of the reaction
mixture after 15 hours, and
the third curve corresponds to the injection of the reaction
mixture after 15 hours+30 minutes of cyclization.
Purification of the glycopeptide
After the synthesis described above, one proceeds to the
purification in two steps:
by size exclusion on a Trisacryl GF05 (100.times.2.3 cm) column
with a flow rate of 10 ml/h, eluted by an aqueous solution
containing 0.1 M acetic acid and 3% n-butanol; this first step
allows the elimination of the non-reacted oligosaccharides as well
as the excess BOP=hexafluorophosphate od
benzotriazolyl-oxy-tris(dimethylamino)phosphonium, and the Glu
pNA,
by ethanolic precipitation (90%), during which the sample is
maintained at 4.degree. C. for 24 h; this second step allows the
elimination of the excess imidazole.
The purification is followed by HPAE-PAD.
In FIG. 2, the fourth curve corresponds to the injection of the
product Lewis.sup.A /Lewis.sup.X -pyroGlu-pNA purified by size
exclusion followed by an ethanolic precipitation (t.sub.r :
17.4/17.6).
Characterization of the glycopeptide (Lewis.sup.A /Lewis.sup.X
-pyroGlu-pNA)
An analysis in .sup.1 H RMN at 300 MHz was conducted.
The Lewis.sup.A /Lewis.sup.X -pyroGlu-pNA was dissolved in D.sub.2
O (6.10.sup.-3 mole/l). Lewis.sup.A possesses a terminal galactose
bound in 3 and a fucose terminal bound in 4 on the
N-acetylglucosamine. Lewis.sup.X possesses a galactose terminal
bound in 4 and a fucose terminal bound in 3 on the
N-acetylglucosamine. Spectrum analysis allowed the identification
of the characteristic protons:
common to the 2 glycopeptides: 8.33 and 7.79 (4H, 2d, H aromatic);
4.90 (2H, m, H5 .alpha.Fuc); 4.67 (1H, d, J.sub.1.2, 7.32 Hz, H1
.beta.GlcNAc); 4.37 (1H, d, J.sub.1,2 7.42 Hz H1
.beta.Gal.sup.int); 4.16 (1H, s, H4 .beta.Gal.sup.int); 2.83 (2H,
m, .gamma.CH.sub.2 pyroGlu); 2.33 (2H, m, .beta. and
.beta.'CH.sub.2 pyroGlu); 2.05 and 2.04 (6H, 2s, CH.sub.3 Ac
GIcNAc); 1.22 and 1.21 (6H, 2s, CH.sub.3 Fuc);
specific of Lewis.sup.A : 5.05 (1H, d, H1 .alpha.Fuc); 4.53 (1H, d,
H1 .beta.Gal);
specific of Lewis.sup.X : 5.24 (1H, d, J.sub.1.2, 7.2 Hz, H1
.alpha.Fuc); 4.50 (1H, d, H1 .beta.Gal).
Example 13
Lewis.sup.B -pyroGlu-pNA and oligoH-pyroGlu-pNA have been prepared
as indicated in example 12.
As reviewed hereafter, the analysis of glycopeptides obtained
includes that of .beta.-lactosyl-pyroGlu-pNA
(N-lactosyl.beta.-pyroGlu-pNA) and Lewis.sup.A /Lewis.sup.X
-pyroGlu-pNA.
Analysis of glycopeptides (Dionex apparatus)
Separation by anion exchange chromatography. Column CarboPac PA1
(4.times.250 mm) Flow rate: 1 ml/min. Detection by pulse
amperometry. Work done at room temperature.
For good separation, a sodium acetate gradient is used:
Time NaOH NaOH 100 mM (min) 100 mM CH.sub.3 COONa 1M 0 100 0
Injection 0,1 100 0 5 100 0 15 80 20 30 0 100 35 0 100 40 100 0
Retention times expressed in minutes:
Imidazole 4.4 .+-. 0.1 Lactose 10.0 .+-. 0.1
.beta.-lactosyl-Glu-pNA 27.5 .+-. 0.1 .beta.-lactosyl-pyroGlu-pNA
22.9 .+-. 0.1 Imidazole 4.5 .+-. 0.1 Lewis.sup.A /Lewis.sup.X
8.9-9.4 .+-. 0.1 Lewis.sup.A /Lewis.sup.X -Glu-pNA 22.5/22.7 .+-.
0.1 Lewis.sup.A /Lewis.sup.X -pyroGlu-pNA 17.4/17.6 .+-. 0.1
Imidazole 4.4 .+-. 0.1 Lewis.sup.B 7.3 .+-. 0.1 Lewis.sup.B
-Glu-pNA 19.7 .+-. 0.1 Lewis.sup.B -pyroGlu-pNA 16.6 .+-. 0.1
Imidazole 4.5 .+-. 0.1 OligoH 8.2/8.7 .+-. 0.1 OligoH-Glu-pNA 23.8
.+-. 0.1 OligoH-pyroGlu-pNA 18.9 .+-. 0.1
* * * * *